Polysaccharides containing carboxylic groups are widely used in several branches like pharmaceutics, medicine, cosmetics, and food. Hyaluronic acid, gellan, xanthan exemplify these products. The fact that many of these products can be recovered from natural sources and that they are biocompatible and biodegradable have promoted their use as a replacement of the conventional synthetic polymers like polyacrylic and polyolefinic products. Among the carboxylated polysaccharides, hyaluronic acid is particularly worthy of note. This polysaccharide is ubiquitous in any animal species and in the unicellular organisms. It is a basic component of the animal connective tissue, it is found in the synovial fluid, in the vitreous humour and in the umbilical cord; it is biocompatible, bioadsorbable, non immunogenic and it carries out an important role in several biological functions (tissue hydration, organisation of the proteoglycans in cartilage, tissue repair, embriogenesis, lubrication and cartilage protection). It is currently used in the treatment of articular diseases, in viscosurgery, as a drug carrier and in the cosmetic field.
The properties of carboxylated polysaccharides can be widely varied by introducing chemical modifications for example at the hydroxyl groups.
Because of the important biological activity of carboxylated polysaccharides, it is very interesting to obtain these polysaccharides in their derivatized forms, thereby affecting their biofunctional features.
An important modification site is the primary hydroxyl group bound to the carbon atom on position 6 of the glicopyranosidic residue.
As an example, the selective halogenation of polysaccharides such as amylose (Macromolecules, 1994, 27, 2917-2922), laminaran (Carb. Res., 1996, 292, 39-46), cyclodextrin (JP08005623), chitosan (J. Carb. Chem., 1997) is well known. Some authors report that they have obtained halogenated derivatives from aminosaccharides or disaccharides such as 2-amino-2-deoxy-glucopyranose (Tetrahedron Left., 1991, 32, 3977-3980), fructofuranose (Carb. Res., 1994, 265, 249-269), cellobiose (Austr. J. Chem., 1997, 50, 13-18).
In polysaccharides containing carboxylic groups, the high reactivity of the carboxylic group, the presence of hydrogen bonds with the solvation water which can lead to the formation of secondary structures, and the high polarity of the group renders difficult to obtain selectively modified derivatives possible. Differently from the neutral polysaccharides.
The Applicant has set up a process for the preparation of carboxylated polysaccharides halogenated on the carbon atom on position 6. The process herein described allows the obtainment of selectively modified derivatives with a high yield and high degree of substitution.
The present invention concerns the field of polysaccharides containing carboxylic groups and hydroxymethylenic groups (i.e. primary hydroxyl groups) . The invention relates to new polysaccharides halogenated on the carbon atom on position 6 of the glicopyranosidic residue and the process for their synthesis.
A new class of polysaccharides containing carboxylic groups selectively substituted with halogens on the carbon atom on position 6 of the glicopyranosidic residue and the procedure to obtain these derivatives are therein described.
The procedure entails the halogenation of carboxylated polysaccharides with alkyl aryl sulfonyl-halides in organic solvent. The products of this invention can be used as substrates in synthesis where the reactive site is the carbon that binds the halogen. Moreover, they can be used in the medical-diagnostic field.
The object of the present invention consists in a group of new derivatives of carboxylated polysaccharides that are selectively substituted with an halogen on the carbon atom on position 6 of the glycopyranosidic residue.
The invention relates to the 6-halo-6-deoxy derivatives of carboxylated polysaccharides where the substituent on position 6 is selected in the group consisting of chlorine, fluorine, bromine, iodine and where the carboxylic group is present either in a free or salified form.
For the purpose of the present invention, the term xe2x80x9ccarboxylated polysaccharidexe2x80x9d indicates each polysaccharide containing primary hydroxyl groups on the carbon atom on position 6 of the glicopyranosidic residue and one or more carboxylic groups.
The polysaccharide can be linear or branched with a molecular weight ranging preferably from 1,000 to 2,000,000, or more preferably from 100,000 to 200,000. Hyaluronic acid, gellan, xanthan, succinoglycan, pectin, chondroitine sulphate, heparan sulphate, dermatan are typical examples of these carboxylated polysaccharides. Hyaluronic acid is the preferred one.
The carboxylated polysaccharide is present either as a free acid or in salified form. The salts are selected in the group consisting of alkaline metals, alkaline earth metals and ammonium salts.
The degree of halogen substitution of these products can range from  greater than 0 to 1, preferably from 0.5 to 1, even more preferably from 0.7 to 0.9.
The term xe2x80x9cdegree of substitutionxe2x80x9d indicates the number of moles of halogen per moles of monosaccharide unit containing an hydroxymethylenic group on position 5 (primary hydroxyl group on position 6). The degree of substitution 1 corresponds to the product having all the hydroxyls on position 6 substituted with the halogen. Examples of products according to the present invention are the 6-deoxy-6 halo derivatives of hyaluronic acid, i.e. the 6-fluoro, 6-chloro, 6-bromo, 6-iododerivatives.
Thanks to the fact that the halogen group can easily be substituted with different groups (a property that is due to the remarkable ability of the halogen group to behave as a leaving group), the halogenated polysaccharides that are the object of the present invention can be used as substrates in the synthesis of 6-substituted carboxylated polysaccharides, where the halogen is substituted by a group suitably selected according to the polarity and hydro/lypophilic properties of the desired final product.
Modifications carried out in this way lead to polysaccharides with physico-chemical features that are modified with respect to the starting product, specifically in as much as biodegradability, bioavailability, ionic strength, thickening/gelling properties are concerned. The 6-chloro, and 6-bromo derivatives are particularly preferred.
Moreover, the products described according to the present invention can be used in the medical-diagnostic field as a contrast medium (nuclear magnetic resonance, tomography, scintigraphy). 6-fluoro and 6-iodio derivatives of hyaluronic acid are preferred.
A further object of the present invention is the process to obtain the carboxylated 6-halo-6-deoxypolysaccharides described above.
The procedure is characterised by the following steps:
a) solvation of the carboxylated polysaccharide, used in free or salified form;
b) halogenation of the solvated polysaccharide with an alkyl or aryl halide in the presence of an organic solvent;
c) possible alkalinization of the system obtained in step b) up to a pH ranging from 9 to 11;
d) neutralization of the system obtained in step b) or c), and recovery of the 6-halo-6-deoxy derivative of the starting carboxylated polysaccharide.
The starting polysaccharide has the features indicated above with reference to the xe2x80x9ccarboxylated polysaccharidexe2x80x9d.
Specifically, it is possible to use, as starting products, polysaccharides in the form of salts with nitrogen-containing heterocycles selected in the group consisting of pyridine, pyrazine, pyrimidine, pyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3,4-tetrazole, 1,2,4,5-tetrazole, 1,2,3,5-tetrazole and their products of partial or total hydrogenation, possibly substituted with one or more alkyl groups containing from 1 to 6 carbon atoms. A specific example of these heterocycles is sym-collidine (2,4,6-trimethylpyridine).
According to the present invention, it is fundamental that the starting polysaccharide undergoes solvation by an organic solvent. By this process, it is possible to obtain the necessary reactivity of the polysaccharide by improving specifically its ability to interact with the halogenating reagent.
Solvation is carried out by means of a prolonged mixing of the polysaccharide with the solvent. The solvent can be selected in the group consisting of dimethylformamide, formamide, dimethylacetamide, preferably dimethylformamide.
In a preferred form, the polysaccharide can be mixed with the organic solvent under stirring for 10 hours. The solvent is further removed by evaporation under reduced pressure.
The solvation can be carried out in a cyclic way; in this case, at the end of the first cycle some fresh solvent is added, mixed again and the solvent is then removed. The procedure can be repeated one or more times. An organic solvent, preferably dimethylformamide, is added to the polysaccharide prepared in this way up to complete dissolution.
An excess of solvent is used for poorly soluble products. The halogenation reaction is carried out by adding an alkyl or aryl sulphonyl halide to the solution. The preferred halides are methanesulphonyl chloride (mesyl chloride), p-toluensulphonyl chloride, methanesulphonyl bromide (mesyl bromide), p-toluensulphonyl bromide.
If non-salified polysaccharides are used as a starting product, the halide is dissolved in a solvent, preferably dimethylformamide and then added to the polysaccharidic solution.
In a preferred embodiment, the halide is added at a temperature ranging from xe2x88x9260xc2x0 C. and xe2x88x9220xc2x0 C., under stirring and under nitrogen flux for not less than one hour. Ten moles of halide per mole of monosaccharide to be halogenated are preferably used.
After addition of the halide, the system is kept under these conditions for a time ranging from 30xe2x80x2 and 2 h; later on, at room temperature for a time ranging from 30xe2x80x2 and 6 hours and finally at 80-100xc2x0 C. for a time ranging from 3 to 72 hours.
If the starting polysaccharide is used in a free form or salified with alkaline or earth alkaline metals, the pH of the mixture obtained after b) is adjusted to a value ranging from 9 to 11; the mixture is then kept for a time ranging preferably from 5 to 20 minutes at room temperature. This step is commonly performed by adding NaOH.
If the starting polysaccharide is used in the form of salt of a nitrogen-containing heterocycle, the solution after halogenation can be directly treated according to step d., i.e. without performing the alkalinization according to step c). The system obtained from b) or c) is then neutralized; the halogenated polysaccharide is recovered from the solution by means of known techniques such as precipitation, drying or freeze drying.
The reaction described herein allows the obtainment of carboxylated polysaccharides whose primary hydroxyl groups on position 6 are substituted with halogen atoms, in a high yield (higher than 80%) and with a high degree of substitution.
The invention is now described in a non limitative way by the following experimental examples.